The failure of HIV-1 vaccines to protect against infection is based in part on their inability to induce a broadly neutralizing antibody (bnAb) response. It is now clear that antibodies directed against the most immunogenic domains (e.g., V3, CD4-bd or CD4-i epitopes) have limited activities against typical clinical isolates, primarily due to effective masking mechanisms, while other conserved targets (e.g., b12, MPER, 2G12) are not immunogenic. Recent studies in our own and other labs have identified the existence of a previously unrecognized class of conserved epitopes that possess potent neutralizing activities for primary isolates, including those in which the classical epitopes are effectively masked. These antibodies are directed against quaternary neutralization epitopes (QNEs) that are dependent on the maintenance of the native trimeric Env structure and are not retained in soluble Env proteins. QNE-specific antibodies frequently possess remarkably potent neutralizing activities, and some have great breadth as well. For example, we have identified an acutely infected patient (CAP256) who after superinfection developed broad and potent bnAbs for subtype C isolates that are directed against QNEs. Despite the broad distribution of these epitopes, antibodies of this sort are rare, suggesting that these quaternary epitopes are usually not very immunogenic. The overall goal of this program is to learn how to reproducibly induce such bnAbs, initially by infection and eventually by vaccination. We will identify additional patients in this cohort that possess similar neutralizing activities, and identify Env variants and epitopes in CAP256 and other patients that may have played a role in inducing these antibodies (Morris/Williamson). The structure and sequence determinants of these epitopes will be defined, and they will be inserted into functional SHIVs (Pinter). Monkeys will then be infected with these SHIVs and the resulting immune responses will be monitored for neutralization breadth and QNE specificity (Hu). The requirement of infection by multiple sequences for induction of such bnAbs will also be explored. MAbs will be isolated via neutralization screens from humans and monkeys producing bnAbs (Robinson) and their epitope specificities, breadth and neutralization potencies characterized. These studies will be supported by Administrative (Pinter), Viral Immunology (Krachmarov) and Molecular Biology (de Parseval) cores. RELEVANCE: Current HIV-1 vaccines have failed, in part due to their inability to induce a broadly neutralizing antibody response. Recent information suggests an important role for a new class of antibodies, directed against broadly conserved epitopes dependent on native quaternary Env structures (QNEs). The overall goal of this program is to develop a strategy for efficiently inducing such broadly protective antibodies. It is hoped that vaccines that are capable of inducing such activities will protect against infection. PROJECT 1: Title: Characterization of Sequence Specificities and Structures of QNEs Project Leader: PINTER, A PROJECT 1 DESCRIPTION (provided by applicant): Key goals of Project 1 will be to map the sequence determinants of known QNEs and novel QNEs that will be identified in studies in Project 2, to define the underlying structure of the Env protein required for QNE formation and expression, to generate these structures as soluble proteins, and to generate chimeric and mutant SHIVs that express QNEs for Infectivity and immunogenicity studies in monkeys (Project 4). The Specific Aims of this Project are: 1- To define minimal sequence determinants for mAbs and pAbs recognizing diverse QNEs, including available antibodies and new reagents that will be identified during this program. The critical determinants in VIV2, V3 and other regions of Env for expression of these epitopes will be defined. 2- To characterize the overall structure and biochemical properties of various QNEs. These studies will address whether QNEs are formed across adjacent protomers in the trimer or in individual subunits in a trimer-dependent manner. The potential correlation between expression of 'masked' Env conformations and formation of QNEs will be determined. 3- To express soluble forms of various QNEs. The ability of artificially stabilized gp140 trimers to express model QNEs recognized by 2909 and PG09 mAbs will be tested by binding assays and neutralization adsorption assays. The effect of stabilizing native trimeric Env structures by introducing inter-subunit disulfide bonds (SOS constructs) on QNE expression will be examined, and if these stabilized constructs express QNEs, soluble forms of the stabilized trimers will be generated. Attempts will also be made to generate monomeric molecules that express these epitopes by mutations that result in reorganization of gp120 structure and by introducing the minimal V2 and V3 determinants into an artificial scaffold that allows these regions to interact under various permutations. 4- To introduce conserved QNEs epitope into infectious SHIV constructs. The sensitivity of a standard pathogenic molecularly cloned SHIV, 1157ipd3, to neutralization by CAP256 serum and mAbs PG09 and PG16 will be tested. If these SHIVs do not express the epitopes, then they will be introduced by small fragment exchanges and site-specific mutagenesis, using information obtained in Aim 1. The resulting chimeric SHIVs will be tested for ability to replicate in monkey PBMCs and for neutralization sensitivity to QNE-specific antibodies. Constructs that possess these activities will be selected for passage in rhesus macaques in Project 3. RELEVANCE: This Project will provide essential information about the sequence determinants and structure of various types of QNEs, and also generate reagents needed by Projects 2 and 3 of this HIVRAD. The products will include soluble antigens that will be used for screening for related antibodies (Projects 2 and 4) and for immunization (Project 3), chimeric and mutant Envs to be used for epitope characterization (Projects 2 and 4), and infectious SHIVs that express QNEs for infectivity and immunogenicity studies in monkeys (Project 3).